Abstract:

Despite the progress in understanding nuclear envelope (NE) reformation
after mitosis, it has remained unclear what drives the required membrane
fusion and how exactly this is coordinated with nuclear pore complex
(NPC) assembly. Here, we show that, like other intracellular fusion
reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE
proteins that require activation by NSF. Antibodies against Xenopus NSF,
depletion of NSF or the dominant-negative NSFE329Q variant specifically
inhibited NE formation. Staging experiments further revealed that NSF
was required until sealing of the envelope was completed. Moreover,
excess exogenous alpha-SNAP that blocks SNARE function prevented
membrane fusion and caused accumulation of non-flattened vesicles on the
chromatin surface. Under these conditions, the nucleoporins Nup107 and
gp210 were fully recruited, whereas assembly of FxFG-repeat-containing
nucleoporins was blocked. Together, we define NSF- and SNARE-mediated
membrane fusion events as essential steps during NE formation downstream
of Nup107 recruitment, and upstream of membrane flattening and
completion of NPC assembly.